Compositions and methods for the treatment and prevention of infections caused by staphylococcus aureus bacteria

ABSTRACT

The present invention relates to antimicrobial deoxyribonuclease-based compositions that inhibit growth and proliferation of  Staphylococcus aureus  bacteria. The present invention also relates to methods of administering the compositions in the treatment and prevention of  S. aureus  infections. The present invention also relates to methods of administering the compositions in the eradication of  S. aureus  nasal carriage, in order to prevent the transmission of  S. aureus  bacteria

RELATIONSHIP TO PRIOR APPLICATIONS

This application claims priority under 35 U.S.C. § 119(e) from U.S.Provisional application No. 60/994,471, filed Oct. 18, 2007

GOVERNMENTAL SUPPORT

The Research leading to the present invention was supported in part, byNational Institutes of Health Grant No. 5R01DE015124. Accordingly, theU.S. Government has certain rights in this invention.

FIELD OF THE INVENTION

The present invention relates to antimicrobial deoxyribonuclease-basedcompositions that inhibit growth and proliferation of Staphylococcusaureus bacteria. The present invention also relates to methods ofadministering the compositions in the treatment and prevention of S.aureus infections. The present invention also relates to methods ofadministering the compositions in the eradication of S. aureus nasalcarriage, in order to prevent the transmission of S. aureus bacteria.

INTRODUCTION

The Gram-positive bacterium Staphylococcus aureus is a major humanpathogen (Lowy, 1998. New Engl. J. Med. 339:520-532). S. aureus causesnumerous infections including acute skin abscesses (pimples, boils,styes, furunculosis) and invasive infections (pneumonia, mastitis,phlebitis, meningitis, urinary tract infections, osteomyelitis), as wellas life-threatening bacteremias and endocarditis. S. aureus is a majorpathogen in nosocomial infections, and in infections in patients withindwelling medical devices. S. aureus is also a major pathogen ininfections of wounds, including infected diabetic foot ulcers, as wellas in burn wounds. S. aureus can also cause toxin-mediated infectionsincluding food poisoning and toxic shock syndrome. Over the past 20years, the frequencies of both nosocomial and community-acquired S.aureus infections has been steadily increasing (Stevens, 2003. Curr.Opin. Infect. Dis. 16:189-191). In addition, numerousmultidrug-resistant strains of S. aureus have emerged in recent years(Bal & Gould, 2005. Expert Opin. Pharmacother. 6:2257-2269). Theseinclude methicillin-resistant S. aureus (MRSA), which are resistant toall penicillinase-resistant penicillins and cephalosporins (Lowy, 1998.New Engl. J. Med. 339:520-532). Infections caused by MRSA are commonlytreated with vancomycin (Pope & Roecker, 2007. Expert Opin.Pharmacother. 8:1245-1261). Recently, however, vancomycin-resistant S.aureus (VRSA) strains have been isolated (Whitener et al., 2004. Clin.Infect. Dis. 38:1049-1055). In addition, S. aureus strains that exhibitresistance to intermediate levels of vancomycin (vancomycin-intermediateS. aureus or VISA) have been isolated (Centers for Disease Control andPrevention, 1997. MMWR Morb. Mortal. Wkly. Rep. 46:624-626). Thepercentage of S. aureus infections caused by MRSA, VRSA and VISA strainshas been increasing (Lodise & McKinnon, 2007. Pharmacother.27:2002-2012). Infections caused by MRSA, VRSA and VISA strains areoften more severe, more easily transmitted, and more difficult to treat,than are infection caused by methicillin-sensitive S. aureus (MSSA)strains (Tristan et al., 2007. J. Hosp. Infect. 65 Suppl 2:105-109).Also, multidrug-resistance may eventually lead to the evolution of S.aureus strains that are resistant to all known antibiotics. New methodsfor treating and preventing S. aureus infections are urgently needed.

S. aureus is the leading cause of hospital-acquired infections. Thefederal Centers for Disease Control and Prevention estimates that in2006 one in 22 hospitalized patients will experience a hospital-acquiredinfection, resulting in a total of 1.7 million infections and 99,000deaths (Sack, 2007. New York Times July 27, p. 1). These nosocomialinfections account for a significant portion of healthcare expendituresin the United States (Lodise & McKinnon, 2007. Pharmacother.27:2002-2012). People who are at a higher risk for S. aureus infectionsinclude hospitalized patients, older patients, patients with type 1diabetes, intravenous drug users, patients undergoing hemodialysis,surgical patients, HIV patients, patients with intravascular devices,patients with prosthetic heart valves, patients taking immunosuppressivedrugs, and patients with defective leukocyte function. The large numberof susceptible patients and the high number of nosocomial infections anddeaths underscores the need for improved methods for treating andpreventing S. aureus infections.

S. aureus is a natural commensal bacterium that colonizes the anteriornares of approximately 30 to 50 percent of healthy adults. Infectionresults when a breach in the mucosal barrier or skin allows bacterialcells access to the underlying tissues or to the bloodstream (Lowy,1998. New Engl. J. Med. 339:520-532). Sites of infection are usuallycolonized by bacteria from the patient's own nasal reservoir, fromcontact with an infected patient, or from exposure to thetransiently-colonized hands of healthcare workers. Previous studies haveshown that eradication of S. aureus nasal carriage results in a decreasein the rate of S. aureus nosocomial infections (Kallen et al., 2005.Infect. Control Hosp. Epidemiol. 26:916-922). Mupirocin cream, appliedtopically to the nares, has been shown to effectively reduce S. aureusnasal carriage (Bertino, 1997. Amer. J. Health Systems Pharm.54:2185-2191). However, mupirocin cream needs to be administered 3 timesper day for 5 days, and mupirocin-resistant MSSA and MRSA strains havebeen identified (Kresken et al., 2004. Int. J. Antimicrob. Agents23:577-581; Hurdle et al., 2005. J. Antimicrob. Chemother.56:1166-1168). Therefore, there is a need for a method for eradicatingS. aureus nasal carriage that is more efficient and less susceptible tothe evolution of antimicrobial resistance.

S. aureus is known for its ability to form biofilms, which are definedas communities of bacteria, encased in a self-synthesized extracellularpolymeric matrix, growing attached to a biotic or abiotic surface (Götz,2002. Mol. Microbiol. 43:1367-1378). Evidence suggests that biofilmformation plays a role in S. aureus wound infections (Akiyama et al.,1996. J. Dermatol. Sci. 11:234-238) and osteomyelitis (Buxton et al.,1987. J. Infect. Dis. 156:942-946). Biofilm formation may also play arole in other localized S. aureus infections. Biofilms that form ontissues or medical devices are extremely difficult to eradicate becausethe biofilm mode of growth protects bacterial cells from killing byantibiotics and host defenses (Fux et al., 2005. Trends Microbiol.13:34-40). Therefore, there is a need for anti-infective therapies thatcan disperse S. aureus biofilms and kill biofilm-embedded S. aureusbacteria.

SUMMARY OF THE INVENTION

In one embodiment, the present invention provides a composition forpreventing and/or inhibiting the growth of biofilm-embedded S. aureusbacteria comprising: (a) a first compound comprising adeoxyribonuclease, or an active fragment or variant thereof, thatdisperses a biofilm; and (b) a second compound comprising anantimicrobial agent that is active against S. aureus cells.

In another embodiment, the deoxyribonuclease enzyme is deoxyribonucleaseI.

In another embodiment, the deoxyribonuclease enzyme is bovinedeoxyribonuclease I.

In another embodiment, the deoxyribonuclease enzyme is humandeoxyribonuclease I.

In yet another embodiment, the antimicrobial agent is the quaternaryammonium compound cetylpyridinium chloride, also known ashexadecylpyridinium chloride.

An embodiment of the invention includes a method for treating a S.aureus infection by administering a composition comprising (a) adeoxyribonuclease enzyme, or a deoxyribonuclease fragment or variantthereof; and (b) an antimicrobial agent or mixture of antimicrobialagents.

In yet another embodiment, the deoxyribonuclease-based antimicrobialcomposition of the invention can be used to treat various kinds ofwounds, including, but not limited to, surgical wounds, accidentalwounds, burn wounds, leg ulcers, foot ulcers, venous ulcers, diabeticulcers, and pressure ulcers.

In yet another embodiment, the deoxyribonuclease-based antimicrobialcomposition of the invention can be used to eradicate S. aureus nasalcarriage.

In yet another embodiment, the deoxyribonuclease-based antimicrobialcomposition of the invention can be used to treat ocular infections.

In yet another embodiment, the deoxyribonuclease-based antimicrobialcomposition of the invention can be used as an antiseptic rinse for useon skin, medical devices, surgical instruments, and the like, before,during or after invasive procedures such as catheter placement orsurgery.

One aspect of the present invention includes providing methods of usingthe deoxyribonuclease-based antimicrobial composition of the inventionin wound care devices, including, but not limited to, a sprayapplicator.

An additional aspect of the present invention includes wound careointments, gels, and lotions comprising the deoxyribonuclease-basedantimicrobial compositions of the invention, in addition to binders,wetting agents, adherents, thickeners, stabilizers, fillers, and thelike.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows that treatment of 24-h-old S. aureus biofilms grown inmicrotiter plate wells with a solution of 100 μg/ml of deoxyribonucleaseI (10 min treatment) causes significant detachment of the biofilm, asjudged by visual inspection of the amount of crystal violet stainingmaterial remaining in the well after treatment.

FIG. 2 shows that the detachment of 24-h-old S. aureus biofilms grown inmicrotiter plate wells by deoxyribonuclease I (10 min treatment) isdependent on the deoxyribonuclease I concentration, as judged byquantitation of the amount of crystal violet stain remaining in the wellafter treatment (Absorbance at 595 nm).

FIG. 3 shows that a solution of 100 μg/ml of deoxyribonuclease I (10 mintreatment) is capable of detaching 5-h-old, 8-h-old, 12-h-old and24-h-old S. aureus biofilms grown in microtiter plate wells.

FIG. 4 shows that S. aureus cells grown in medium supplemented with 100μg/ml of deoxyribonuclease I exhibit much less clumping(autoaggregation) than do cells grown in unsupplemented medium.

FIG. 5 shows that S. aureus cells grown in tubes in medium supplementedwith 100 μg/ml of deoxyribonuclease I exhibit much less biofilmformation than do cells grown in unsupplemented medium.

FIG. 6 shows that S. aureus cells grown in microplate wells in mediumsupplemented with 100 μg/ml of deoxyribonuclease I are incapable offorming distinct biofilm colonies.

FIG. 7 shows that the inhibition of S. aureus biofilms grown inmicrotiter plate wells by deoxyribonuclease I is dependent on thedeoxyribonuclease I concentration.

FIG. 8 shows that S. aureus biofilm cells grown in microplate wells areresistant to killing by 100 μg/ml of deoxyribonuclease I for 10 min, andby 0.3% cetylpyridinium chloride (CPC) for 5 min, but that treatment ofthe biofilms with 100 μg/ml of deoxyribonuclease I for 10 min followedby treatment with 0.3% CPC for 5 min results in significant killing ofthe biofilm cells.

FIG. 9 shows that treatment of S. aureus biofilm cells grown inmicroplate wells with 100 μg/ml of deoxyribonuclease I for 10 minfollowed by treatment with 0.3% CPC for 3 min or 5 min results insignificant killing of the biofilm cells.

DETAILED DESCRIPTION OF THE INVENTION

This invention relates to a method and composition for preventing and/orinhibiting the growth of biofilm-embedded S. aureus bacteria. The basisof the invention is the discovery that the bacteria is most susceptiblewhen it is subject first to a means of detaching S. aureus biofilm andthen is exposed to an agent which kills the bacteria.

It has been found that deoxyribonuclease enzyme or active fragment orvariant thereof is capable of inhibiting S. aureus biofilm formationwhen added to a culture medium. Examples include human deoxyribonucleaseI and bovine deoxyribonuclease I.

Agents which are capable of killing S. aureus are known in the art andinclude antimicrobial compounds such as quaternary ammonium salts.Examples of quaternary ammonium salts include for example, but notlimited to, cetylpyridinium chloride, methacryloyloxydodecylpyridinimium bromide, like pyridinium halide salts,benzalkoniumchloride, methacryloxylethylbenzyl dimethylammonium chlorideand methacryloxylethylcetyldimethyl ammonium chloride.

The S. aureus can be treated by the administration of thedeoxyribonuclease enzyme and the antimicrobialanat agent at the sametime or serially with the deoxyribonuclease enzyme being administeredbefore the antimicrobial agent.

Any pharmaceutically acceptable vehicle or carrier, as well as adjuvant,can be used in the manufacture, dissolution and administration ofpharmaceutical preparations of the invention comprisingdeoxyribonuclease enzyme or active fragment or variant thereof and/orthe antimicrobial agent. Such vehicles, carriers and adjuvants are wellknown to those of skill in the art and described in text books such asRemington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa.,1985. Appropriate concentrations of active composition to beincorporated into pharmaceutical compositions can be routinelydetermined by those skilled in the art and is dependent upon the form ofadministration as well as the severity of the condition being treated.

Pharmaceutical formulations suitable for oral administration may beprovided in convenient unit forms including, but not limited to,capsules or tablets, each containing a predetermined amount of thedeoxyribonuclease enzyme or active fragment or variant thereof and/orthe antimicrobial agent; as a powder or granules; as a solution, asuspension or as an emulsion. The deoxyribonuclease enzyme or activefragment or variant thereof and/or the antimicrobial agent can also bepresented as a bolus, electuary, or paste. Tablets and capsules for oraladministration may contain conventional excipients such as bindingagents, fillers, lubricants, disintegrants, or wetting agents. Thetablets may be coated according to methods well known in the art. Timedrelease formulations, which are known in the art, may also be suitable.Oral liquid preparations may be in the form of, for example, aqueous oroily suspensions, solutions, emulsions, syrups or elixirs, or may bepresented as a dry product for constitution with water or other suitablevehicles before use. Such liquid preparations may contain conventionaladditives such as suspending agents, non-aqueous vehicles, includingedible oils, or preservatives.

Deoxyribonuclease enzyme or active fragment or variant thereof and/orthe antimicrobial agent of the present invention may also be formulatedfor parenteral administration, such as by injection, for example bolusinjection or continuous infusion, and may be provided in unit dose formin ampules, pre-filled syringes, small volume infusion or in multi-dosecontainers with an added preservative. Pharmaceutically acceptablecompositions comprising a deoxyribonuclease enzyme or active fragment orvariant thereof and/or the antimicrobial agent for parenteraladministration may be in the form of a suspension, solution or emulsionin oily or aqueous vehicles, and may contain formulatory agents such assuspending, stabilizing, and/or dispersing agents. Alternatively, theactive ingredient may be in powder form, obtained by asceptic isolationof sterile solid or by lyophilization from solution, for constitutionwith a suitable vehicle such as sterile, pyrogen free water, before use.

For topical administration to the epidermis, deoxyribonuclease enzyme oractive fragment or variant thereof and/or the antimicrobial agent of thepresent invention may be formulated in an ointment, cream, or lotion, oras a transdermal patch. Ointments and creams, may, for example, beformulated with an aqueous or oily base with the addition of suitablethickening and/or gelling agents. Lotions may be formulated with anaqueous or oily base and will in general also contain one or moreemulsifying agents, stabilizing agents, suspending agents, thickeningagents, or coloring agents. Formulations suitable for topicaladministration in the mouth include lozenges comprisingdeoxyribonuclease enzyme or active fragment or variant thereof and/orthe antimicrobial agent in a flavored base, usually sucrose and acaciaor tragacanth; pastilles comprising the active ingredient in an inertbase such as gelatin and glycerin or sucrose and acacia; and mouthwashes comprising the active ingredient in a suitable liquid carrier.For topical administration to the eye, the deoxyribonuclease enzyme oractive fragment or variant thereof and/or the antimicrobial agent can bemade up in solution or suspension in a suitable sterile aqueous ornon-aqueous vehicle. Additives such as buffers (e.g. sodiummetabisulphite or disodium edeate) and thickening agents such ashypromellose can also be included.

For intra-nasal administration, deoxyribonuclease enzyme or activefragment or variant thereof and/or the antimicrobial agent of thepresent invention can be provide in a liquid spray or dispersible powderor in the form of drops. Drops may be formulated with an aqueous ornon-aqueous base also comprising one or more dispersing agents,solubilizing agents, or suspending agents. Liquid sprays areconveniently delivered from pressurized packs. For administration byinhalation, deoxyribonuclease enzyme or active fragment or variantthereof and/or the antimicrobial agent of the present invention can bedelivered by insufflator, nebulizer or a pressurized pack or otherconvenient means of delivering the aerosol spray. Pressurized packs maycomprise a suitable propellant such as dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide orother suitable gas. In the case of a pressurized aerosol the dosage unitmay be determined by providing a valve to deliver a metered amount.

Alternatively, for administration by inhalation or insufflation, thedeoxyribonuclease enzyme or active fragment or variant thereof and/orthe antimicrobial agent of the present invention can take the form of adry powder composition, for example a powder mix of the active componentand a suitable powder base such as lactose or starch. The powdercomposition may be presented in unit dosage form in, for example,capsules, cartridges or blister packs of gelatins, from which the powdercan be administered with the aid of an inhalator or insufflator.

When desired, any of the above-described formulations may be adapted toprovide sustained release of the deoxyribonuclease enzyme or activefragment or variant thereof and/or the antimicrobial agent.

The amount of deoxyribonuclease enzyme or active fragment or variantthereof and/or the antimicrobial agent of the present invention requiredfor use in treatment will of course vary not only with the particularprotein or active fragment or variant selected but also with the routeof administration, the nature of the condition being treated, and theage and condition of the organism.

Increasing detachment of bacteria from a biofilm is also expected todecrease resistance of the bacteria to antibiotic therapy. Accordingly,the present invention also provide methods for enhancing efficacy ofantibiotic therapy against bacterial infections by administration of apharmaceutical composition of the present invention in combination withor prior to administration of an antibiotic.

In another embodiment of the present invention, wound dressingsincluding but not limited to sponges or gauzes can be impregnated withthe isolated deoxyribonuclease enzyme or active fragment or variantthereof and/or the antimicrobial agent thereof to prevent or inhibitbacterial or fungal attachment and reduce the risk of wound infections.Similarly, catheter shields as well as other materials used to cover acatheter insertion sites can be coated or impregnated with adeoxyribonuclease enzyme or active fragment or variant thereof and/orthe antimicrobial agent to inhibit bacterial or fungal biofilmattachment thereto. Adhesive drapes used to prevent wound infectionduring high risk surgeries can be impregnated with the isolated proteinor active fragment or variant thereof as well. Additional medicaldevices which can be coated with a deoxyribonuclease enzyme or activefragment or variant thereof and/or the antimicrobial agent thereofinclude, but are not limited, central venous catheters, intravascularcatheters, urinary catheters, Hickman catheters, peritoneal dialysiscatheters, endotracheal catheters, mechanical heart valves, cardiacpacemakers, arteriovenous shunts, schleral buckles, prosthetic joints,tympanostomy tubes, tracheostomy tubes, voice prosthetics, penileprosthetics, artificial urinary sphincters, synthetic pubovaginalslings, surgical sutures, bone anchors, bone screws, intraocular lenses,contact lenses, intrauterine devices, aortofemoral grafts and vasculargrafts. Exemplary solutions for impregnating gauzes or sponges, cathetershields and adhesive drapes or coating catheter shields and othermedical devices include, but are not limited to, phosphate bufferedsaline (pH approximately 7.5) and bicarbonate buffer (pH approximately9.0).

In yet another embodiment, an isolated deoxyribonuclease enzyme oractive fragment or variant thereof and/or the anatimicrobial agent canbe incorporated in a liquid disinfecting solution. Such solutions mayfurther comprise antimicrobials or antifungals such as alcohol,providone-iodine solution and antibiotics as well as preservatives.These solutions can be used, for example, as disinfectants of the skinor surrounding area prior to insertion or implantation of a device suchas a catheter, as catheter lock and/or flush solutions, and asantiseptic rinses for any medical device including, but not limited tocatheter components such as needles, Leur-Lok connectors, needlelessconnectors and hubs as well as other implantable devices. Thesesolutions can also be used to coat or disinfect surgical instrumentsincluding, but not limited to, clamps, forceps, scissors, skin hooks,tubing, needles, retractors, scalers, drills, chisels, rasps and saws.

The compositions and method of the invention can be used for thetreatment and prevention of wound and burn infections caused by S.aureus as well as other infections caused by S. aureus including boilsand sties and bovine mastitis. The compositions can be used as apreprocedural rinse for surgery, as an antiseptic rinse, a topicalantiseptic and a catheter lock solution.

The composition and method of the instant invention can also be used forthe treatment and prevention of biofilm infections caused by otherbacteria including otitis media, sinusitis and chronic obstructivepulmonary disease (Haemophilus influenzae), dental caries (Streptococcusmutans), acne (Propionibacterium acnes), and periodontitis(mixed-species biofilms).

EXAMPLES Example 1 Deoxyribonuclease I Causes the Detachment andDispersal of S. aureus Biofilms

S. aureus Strain SH1000 (Horsburgh et al., 2002. J. Bacteriol.184:5457-5467) was used in all of the following examples. The bacteriawere passaged weekly on blood agar and stored at 4° C. Biofilms werecultured in Tryptic Soy broth (Becton-Dickinson, Sparks, Md.) containing6 g of yeast extract and 8 g of glucose per liter (TSB medium). Allcultures were incubated at 37° C.

A biofilm formation assay was carried out as follows. A loopful of cellsfrom an agar plate was transferred to a polypropylene microcentrifugetube containing 200 μl of TSB medium. The cells were crushed with adisposable pellet pestle, vortexed for 30 sec, diluted to 1 ml in freshTSB medium, and then passed through a 5-μm pore-size syringe filter toremove large clumps of cells as previously described (Kaplan & Fine,2002. Appl. Environ. Microbiol. 68:4943-4950). Filtered cells werediluted to 10³-10⁵ CFU/ml in TSB medium. Aliquots of cells (200 μl each)were transferred to the wells of a 96-well tissue-culture-treatedpolystyrene microtiter plate (Falcon no. 324662, Becton-Dickinson) andthe plate was incubated for 24 h. The biofilms were rinsed once withwater and then treated with 200 μl of deoxyribonuclease I (bovinedeoxyribonuclease I, purchased from Sigma Chemical Company) at 100 μg/mlin 150 mM NaCl, 1 mM CaCl₂. Control biofilms were treated with 200 μl of150 mM NaCl, 1 mM CaCl₂ alone. After 10 min at 37° C., biofilms wererinsed with water and then dried. Biofilms were stained for 1 min with200 μl of Gram's crystal violet stain (catalog no. 23255960, FisherScientific, Fair Lawn, N.J.) and then rinsed with water and dried.Previous studies showed that crystal violet stains the bacterial biofilmbiomass but not the polystyrene microplate substrate (O'Toole & Kolter,1998. Mol. Microbiol. 28:449-462).

FIG. 1 shows that the deoxyribonuclease I solution caused the nearlycomplete detachment of the S. aureus biofilm from the microplate wellsurface, as judged by the amount of crystal violet staining materialthat remained in the well after treatment.

FIG. 2 shows the results of a similar experiment, except that increasingamounts of deoxyribonuclease I were used, and the amount of biofilmbiomass remaining in the wells was quantitated by destaining thebiofilms for 10 min with 33% acetic acid (by vol) and then measuring theabsorbance of the crystal violet solution at 595 nm (A₅₉₅).Concentrations of deoxyribonuclease I that were less than 0.1 μg/mlcaused little detachment of the biofilm. Concentrations ofdeoxyribonuclease I that were between 0.1 and 10 μg/ml caused partialdetachment of the biofilm. Concentrations of deoxyribonuclease I thatwere greater than 10 μg/ml caused near complete detachment of thebiofilm.

FIG. 3 shows the results of a similar experiment, except that S. aureusbiofilms that were grown for 5, 8, 12 or 24 h were used. Theconcentration of deoxyribonuclease I was 100 μg/ml and the treatmenttime was 10 min. In this case, the amount of biofilm biomass remainingin the well was quantitated by measuring the A₅₉₅ of the crystal violetstained biomass as described above, and the percent of biofilm celldetachment was calculated using the formula:1−(A₅₉₅[buffer+deoxyribonuclease I]/A₅₉₅[buffer alone])×100. As can beseen in FIG. 3, the deoxyribonuclease I solution caused significantdetachment of all of the S. aureus biofilms, regardless of their age.

Example 2 Deoxyribonuclease I Inhibits S. aureus Autoaggregation andBiofilm Formation

A series of experiments was performed in order to demonstrate thatdeoxyribonuclease I inhibits S. aureus autoaggregation and biofilmformation. These experiments were carried out as described above, exceptthat biofilms were grown in 16-mm×100-mm PET tubes (2 ml culture vol) ina rotary shaker for 16 h. FIG. 4 shows S. aureus SH1000 cells culturedin this manner in unsupplemented TSB medium formed large aggregates,whereas cells cultured in this manner in TSB medium supplemented with100 μg/ml of deoxyribonuclease I formed smaller aggregates. Crystalviolet staining of the culture tubes showed that deoxyribonuclease Iinhibited biofilm formation at the air-liquid interface (FIG. 5).

FIG. 6 shows that SH1000 biofilms grown for 24 h in 96-well microtiterplates in unsupplemented TSB medium formed distinct, spherical coloniesthat were tightly attached to the microwell surface, whereas biofilmsgrown in TSB medium supplemented with 100 μg/ml of deoxyribonuclease Iformed a dense film that uniformly covered the microwell surface, butwhich readily detached after gentle rinsing. FIG. 7 shows thatdeoxyribonuclease I inhibited SH1000 biofilm formation in adose-dependent manner, as determined by measuring the A₅₉₅ of thecrystal violet stained biofilm biomass as described above.

Example 3 Deoxyribonuclease I Increases the Sensitivity of S. aureusBiofilm Cells to Killing by the Quaternary Ammonium CompoundCetylpyridinium Chloride (CPC)

Biofilms were grown for 24 h in 96-well microtiter plates as describedabove. Biofilms were rinsed once with water and then treated with 200 μlof TSB medium containing 100 μg/ml of deoxyribonuclease I. Control wellswere treated with 200 μl of TSB medium alone. After 10 min at 37° C., 20μl of 3% CPC was added to each well and biofilms were incubated for 5min at room temperature. Control wells received 20 μl of water. Forbiofilms treated with TSB medium alone, biofilms were washed four timeswith phosphate buffered saline to remove the CPC, and then treated with100 μg/ml of deoxyribonuclease I to dissolve the biofilm. This reactionswas carried out in 100 μg/ml in 150 mM NaCl, 1 mM CaCl₂ as describedabove. After 10 min, cells were mixed and then serial dilutions wereplated on agar. For S. aureus biofilms treated with deoxyribonuclease I,cells were mixed and then a 50-μl aliquot of cells was diluted in 50 mlof phosphate buffered saline. The cells were passed through ananalytical test filter funnel (no. 145-2020; Nalgene, Rochester, N.Y.),and the filter was rinsed with 250 ml of sterile water, asepticallyremoved from the filter unit, and placed on a blood agar plate. Colonieswere enumerated after 24 h.

As can be seen in FIG. 8, S. aureus biofilms treated with eitherdeoxyribonuclease I alone or CPC alone did not exhibit a significantdecrease in CFU/well values, whereas S. aureus biofilms treated withdeoxyribonuclease I followed by CPC exhibited an approximately4-log-unit decrease in CFU/well values. A significant decrease in theCFU/well values was also observed after a 10 min deoxyribonuclease Itreatment followed by a 3 min CPC treatment (FIG. 9).

1. A composition for preventing and/or inhibiting the growth ofbiofilm-embedded S. aureus bacteria comprising: (a) a first compoundcomprising a deoxyribonuclease, or an active fragment or variantthereof, that disperses a biofilm; and (b) a second compound comprisingan antimicrobial agent that is active against S. aureus cells.
 2. Thecomposition of claim 1 wherein the deoxyribonuclease enzyme isdeoxyribonuclease I.
 3. The composition of claim 1 wherein thedeoxyribonuclease enzyme is bovine deoxyribonuclease I.
 4. Thecomposition of claim 1 wherein the deoxyribonuclease enzyme is humandeoxyribonuclease I.
 5. The composition of claim 1 wherein theantimicrobial agent is a quaternary ammonium compound.
 6. Thecomposition of claim 5 wherein the quaternary ammonium compound iscetylpyridinium chloride.
 7. A method for treating a S. aureus infectionwhich comprises administering a composition comprising (a) adeoxyribonuclease enzyme, or a deoxyribonuclease fragment or variantthereof; and (b) an antimicrobial agent or mixture of antimicrobialagents.